Breast cancer (BC) is the leading cause of cancer mortality in women worldwide. Triple negative breast cancer (TNBC) represents an aggressive subtype with limited therapeutic options that exhibits high rates of recurrence and metastasis. These tumors are called triple negative because they do not express the estrogen and progesterone receptors or overexpress the HER2 oncogene, which makes them refractory to current targeted therapeutic regimens. Although cancer stem cells (CSCs) remain controversial, studies support a role for a self- renewing cell population in tumor maintenance and therapeutic resistance. NANOG is a master regulator of pluripotency and self-renewal that has been implicated in breast cancer progression. Through collaborative efforts of the Lathia and Reizes laboratories, we have developed TNBC cell lines containing a green fluorescent protein (GFP) reporter driven by the NANOG promoter to analyze the stem cell state in real time. Our preliminary data show that cells expressing GFP from the NANOG promoter exhibit cellular characteristics of CSCs, whereas GFP-negative cells do not, suggesting these cells are non-CSCs. We plan to use these reporter cells to interrogate how CSCs communicate within tumors to orchestrate growth and metastasis, and here we focus on the role of CSC cell-cell communication in metastatic progression. Through the analysis of Oncomine datasets, we found that connexins, the protein subunits of gap junctions, one method of cell-cell communication, are differentially expressed in TNBCs and inform patient outcomes. Biochemical analysis of CSCs and non-CSCs using our reporter lines shows selective expression of connexin 26 (Cx26) in CSCs. The translational goal of this project is to yield proof-of-concept evidence for a therapeutic benefit of blocking cell-cell communication in CSC pluripotency and invasiveness/metastasis. Based on our observations, we hypothesize that the inhibition of gap junctions will yield therapeutic benefit against TNBC recurrence and metastasis by blocking CSC self-renewal and viability. We will test this hypothesis through the following two aims. Aim 1 will test the hypothesis that Cx26 is necessary for CSC maintenance and metastasis. Aim 2 will test the therapeutic benefit of gap junction inhibition on CSC maintenance and metastatic progression. The long-term goal of this research is to address the unmet medical need for effective TNBC therapies with a therapeutic focus on nonhormonal and nongenetic approaches to inhibit tumor progression by attenuating cell-cell communication in CSCs.